The immune system of a mammalian organism produces antibodies which are also called immunoglobulins as a response to the introduction of foreign substances. They are used to defend against the foreign substances which are also referred to as antigens. The immunoglobulins can be divided into five different classes. One distinguishes between immunoglobulins of the M, G, A, E, and D classes. These five immunoglobulin classes each differ with respect to the composition of the heavy chain, which is referred to as the μ, γ, α, ε, or δ chain.
Each immunoglobulin class has a different function in the organism. Immunoglobulins of the M class occur when a first contact is made with the antigen, the so-called primary immunization. However, the concentration of these immunoglobulins decreases rapidly as the infection progresses. The immunoglobulins of the G class are firstly slowly formed during a primary immunization and occur in large amounts when there is a second infection with the same antigen. The immunoglobulins of the A class are found on the mucosal surfaces of the organism and are responsible for the defense processes that occur there. The immunoglobulins of the E class are mainly responsible for allergic reactions. The exact function of the immunoglobulins of the D class is hitherto unknown.
The individual immunoglobulin classes occur in blood in very different concentrations. Thus immunoglobulins of the G class (IgG) are the class with the highest occurrence in human serum, being present in a proportion of about 75% which corresponds to a serum content of 8 to 18 mg/ml. The second most frequent immunoglobulin is IgA, whose average serum concentration is 0.9 to 4.5 mg/ml. Immunoglobulins of the M class are present at a concentration of 0.6 to 2.8 mg/ml, and immunoglobulins of class D are present at a concentration of 0.003 to 0.4 mg/ml. IgE antibodies are present in the lowest proportion and only occur at a concentration of 0.02 to 0.05 μg/ml in serum.
For the differential diagnostics of many diseases, it is important to detect antibodies of one or more very particular immunoglobulin classes that are specific for a certain antigen. A satisfactory diagnosis in the case of viral, bacterial and parasitic infection can only be ensured by means of a class-specific antibody detection or by excluding the presence of certain immunoglobulin classes (e.g. detection of IgG and IgA antibodies but no detection of IgM antibodies). This is particularly important for differentiating between fresh or acute infections and older infections as well as to clinically monitor the course of an infection. The class-specific detection of antibodies is especially important for HIV, hepatitis A, hepatitis B, toxoplasmosis, rubella and chlamydia infections. The class-specific detection of antibodies that are specific for a certain antigen is also necessary when determining the titre of protecting antibodies and for checking whether an immunization has been successful.
Various methods are described in the prior art for detecting antibodies of a particular class that are specific for an antigen. Thus antigen-specific antibodies of a particular class are often detected by binding the specific antibody to a solid phase coated with the specific antigen. The immunoglobulins (Ig) that are specific for the antigen and are now bound to the solid phase are detected by binding antibodies that are directed specifically against human Ig of a certain class to the Ig molecules to be detected. The antibodies that are directed against human Ig are provided with a label which is used for the detection. However, such a test procedure is only possible when all unspecific, non-bound Ig is removed by washing before the reaction with the class-specific labelled antibodies directed against human Ig. Thus, for example, when detecting specific IgG molecules in a sample, relatively large amounts (4-20 mg/ml) of unspecific IgGs are present which can absorb sample specifically to different extents and bind unspecifically to the solid phase. If a detection antibody against IgGs is used, these unspecifically bound immunoglobulins will also be recognized and bound. This results in elevated background signals and reduced sensitivity.
One method of reducing these background signals is to modify the solid phase in order to avoid unspecific binding of the immunoglobulins and to use special buffer additives which are also intended to prevent binding of immunoglobulins to the solid phase (examples: HydroGel solid phase (Perkin Elmer), FAST Slides (Schleicher & Schüll), detergents, chaotropic salts). The modifications of the solid phase are laborious and expensive. Furthermore, it has emerged that buffer additives can reduce the reactivity of some antibodies and thus reduce the signals. The background signals induced by unspecifically bound immunoglobulins increase the blank value which makes it more difficult to detect specific antibodies of a certain immunoglobulin class, especially in the case of miniaturized test systems such as immunoassays in an array format which comprise a plurality of specific tests, in some cases in different test formats, in a reaction vessel. Thus, for example, addition of a certain detergent can suppress the unspecific binding of antibodies, but the same detergent can have no effect or even the opposite effect in another test on the same array system.
The use of the coagulation factor C1q, which is a subunit of the first complement component, as a further possibility of reducing background signals in immunoassays is disclosed in EP 0222146 B1. The protein C1q bound to a support is in this case used to selectively remove circulating immune complexes in vivo from the blood by means of extracorporeal immune adsorption in which immune complexes bound to the protein C1q are separated from the body fluids by separating the solid phase. In U.S. Pat. No. 5,698,449 A1, a fragment of C1q is disclosed for selectively removing immune complexes from the blood and for detecting and quantifying the immune complexes. In addition U.S. Pat. No. 4,062,935 A1 describes the addition of rheumatoid factors or C1q to the sample and the binding and quantification of the resulting immune complexes. However, the prior art described here does not show any application for immunoassays in an array format. A characteristic feature of immunoassays in an array format is the solid phase. In such methods the solid phase preferably consists of localized test areas which comprise defined, discrete areas of the solid phase and are preferably spatially separated from other test areas by inert areas. These localized test areas that are defined as spots preferably have a diameter of 10 μm to 1 cm and particularly preferably a diameter of 100-200 μm. Solid phases having several test areas which are also referred to as array systems are preferred. Such array systems are described, for example, in Ekins and Chu (Clin. Chem. 37 (1995), 1955-1967) and in U.S. Pat. Nos. 5,432,099, 5,516,635 and 5,126,276. Array systems have the advantage that several analyte determinations can be carried out simultaneously from one sample. Hence it is possible to apply a plurality of binding partners such as antigen-specific antibodies to the test field. The solid phase of these array systems can be preferably coated with streptavidin or avidin as disclosed in EP 0939319 (Hornauer et al.). Sample components and in particular unspecific IgGs can bind to all these solid phases. In this case it is impossible to use a universal buffer additive to reduce the background signals or it is only possible with a large amount of effort since each individual binding partner requires a very particular buffer additive. Buffer additives which have positive effects in the case of one binding partner may even have adverse effects for other binding partners. It is also very difficult to modify the solid phase for numerous different binding partners. Hence it is impossible to use the above-mentioned methods with a practicable amount of effort to optimize the blank value when several to many different tests are combined on an array solid phase.
Hence the object was to develop a method for carrying out an immunoassay for detecting antigen-specific antibodies in an array format which largely avoids the disadvantages of the prior art and in particular reduces the background signals due to unspecifically-bound immunoglobulins.